1,25-(OH)_(2)-VitD3通过抑制Snail1-SMAD3/SMAD4复合物形成减轻糖尿病肾病肾小管间质纤维化  

1,25-(OH)_(2)-VitD3 attenuates renal tubulointerstitial fibrosis in diabetic kidney disease by inhibiting Snail1-SMAD3/SMAD4 complex formation

作　　者：黄成宠 董蓉[2,3] 李正胜 袁静 HUANG Chengchong;DONG Rong;LI Zhengsheng;YUAN Jing(The Second Affilated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550002;Department of Nephrology,Guizhou Provincial People's Hospital,NHC Key Laboratory of Pulmonary Immunological Disease,Guiyang 550002;College of Medicine,Guizhou University,Guiyang 550025;Department of Nephrology,The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550001,China)

机构地区：[1]贵州中医药大学第二临床医学院,贵州贵阳550002 [2]贵州省人民医院肾内科,国家卫生健康委员会肺脏免疫性疾病诊治重点实验室,贵州贵阳550002 [3]贵州大学医学院,贵州贵阳550025 [4]贵州中医药大学第二附属医院肾内科,贵州贵阳550001

出　　处：《细胞与分子免疫学杂志》2023年第4期325-331,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：贵州省科技厅科技计划项目(黔科合基础[2019]1194号);贵州省卫生健康委科学技术基金(gzwjkj2019-1-107);国家自然科学基金(81760134);贵州省肾脏病临床医学研究中心(黔科合平台人才[2020]2201号);2018年贵州省高层次创新人才项目(黔科合平台人才[2018]5636);2019贵州省科技支撑计划项目(黔科合支撑[2019]2801号)

摘　　要：目的研究1,25-(OH)2-VitD3(VitD3)对糖尿病肾病肾小管间质纤维化的影响.方法NRK-52E肾小管上皮细胞分为对照组(5.5 mmol/L葡萄糖培养基处理)、高糖组(25 mmol/L葡萄糖的培养基处理)和高糖加VitD3组(25 mmol/L葡萄糖培养基联合10-8 mol/L VitD3).实时定量PCR和Western blot法分别检测NRK-52E细胞Snail家族转录抑制物1(Snail1)、SMAD家族成员3(SMAD3)、SMAD4、α平滑肌肌动蛋白(α-SMA)和上皮钙黏蛋白(E-cadherin)的mRNA和蛋白表达;免疫荧光细胞化学染色检测Snail1、SMAD3、SMAD4的表达及定位;通过染色质免疫沉淀检测Snail1与SMAD3/SMAD4形成的复合物与柯萨奇病毒-腺病毒受体(CAR)的启动子结合情况;荧光素酶报告检测Snail1、SMAD3/SMAD4和E-cadherin的相互作用;采用小干扰RNA(siRNA)抑制细胞Snail1、SMAD4的表达后,通过实时定量PCR检测E-cadherin的mRNA表达.SD大鼠随机分为对照组、DKD组和VitD3处理组.DKD组和VitD3处理组通过注射链脲佐菌素(STZ)先制备DKD模型,DKD造模成功后,VitD3处理组予60ng/kgVitD3灌胃,对照组和DKD组给予生理盐水灌胃;DKD组和VitD3处理组同时皮下注射胰岛素(1~2)U/kg控制血糖,持续8周.采用实时定量PCR和Western blot法分别检测肾组织中Snail1、SMAD3、SMAD4、α-SMA、E-cadherin的mRNA和蛋白水平,免疫组织化学检测肾组织Snail1、SMAD3、SMAD4、α-SMA和E-cadherin的表达及定位.结果与对照组相比,高糖处理的NRK-52E细胞及DKD肾组织Snail1、SMAD3、SMAD4和α-SMA的mRNA和蛋白表达均上调,E-cadherin表达下调;给予VitD3干预后,DKD模型中Snail1、SMAD3、SMAD4、α-SMA和E-cadherin的表达水平恢复到与对照组接近.染色质免疫沉淀提示Snail1、SMAD3/SMAD4与CAR的启动子Ⅳ结合,而VitD3能够阻止Snail1、SMAD3/SMAD4与CAR的启动子Ⅳ结合;荧光素酶报告检测证实Snail1、SMAD3/SMAD4和E-cadherin的互作关系;用siRNA抑制Snail1、SMAD4的mRNA后,上调高糖诱导下E-cadherin的表达.结论VitD3可抑制SnaObjective To investigate the effect of 1,25-(OH)_(2)-VitD3(VitD3)on renal tubuleinterstitial fibrosis in diabetic kidneydisease.Methods NRK-52E renal tubular epithelial cells were divided into control group(5.5 mmol/L glucose medium treatment),high glucose group(25 mmol/L glucose medium treatment)and high glucose with added VitD3 group(25 mmol/L glucose medium combined with 10^(-8) mmol/L VitD3).The mRNA and protein expression of Snai1,SMAD3,SMAD4,a-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively.The expression and localization of Snail,SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining.The binding of Snaill with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor(CAR)was detected by chromatin immunoprecipitation.The interaction among Snaill,SMAD3/SMAD4 and E-cadherin were detected by luciferase assay.Small interfering RNA(siRNA)was used to inhibit the expression of Snaill and SMAD4,,and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR.SD rats were randomly divided into control group,DKD group and VitD3-treated group.DKD model was established by injection of streptozotocin(STZ)in DKD group and VitD3-treated group.After DKD modeling,VitD3-treated group was given VitD3(60 ng/kg)intragastric administration.Control group and DKD group were given normal saline intragastric administration.In the DKD group and VitD3-treated group,insulin(1-2 U/kg)was injected subcutaneously to control blood glucose for 8 weeks.The mRNA and protein levels of Snail,SMAD3,SMAD4,a-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively.Immunohistochemistry was used to detect the expression and localization of Snaill,SMAD3,SMAD4,α-SMA and E-cadherin in renal tissue.Results Compared with the control group,the mRNA and protein expressions of Snail1,SMAD3,SMAD4 and a-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues wer

关 键 词：1 25-(OH)2-VitD3 Snail1-SMAD3/SMAD4复合物 糖尿病肾病 肾间质纤维化 

分 类 号：R692[医药卫生—泌尿科学] R692.6[医药卫生—外科学] R587.1[医药卫生—临床医学] R392-33